Variants of hepatitis b virus with resistance to anti-viral nucleoside agents and applications thereof

ABSTRACT

The present invention relates generally to viral variants exhibiting reduced sensitivity to particular agents and/or reduced interactivity with immunological reagents. More particularly, the present invention is directed to hepatitis B virus (HBV) variants exhibiting complete or partial resistance to nucleoside or nucleotide analogs or other antagonists of HBV DNA polymerase activity and/or reduced interactivity with antibodies to viral surface components including reduced sensitivity to these antibodies. The present invention further contemplates assays for detecting such viral variants. These assays are useful in monitoring anti-viral therapeutic regimens and in developing new or modified vaccines directed against viral agents and in particular the resistant HBV variants of the present invention. The present invention also contemplates the use of the viral variants to screen for agents capable of inhibiting infection, replication and/or release of the virus.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to viral variants exhibitingreduced sensitivity to particular agents and/or reduced interactivitywith immunological reagents. More particularly, the present invention isdirected to hepatitis B virus (HBV) variants exhibiting complete orpartial resistance to nucleoside or nucleotide analogs or otherantagonists of HBV DNA polymerase activity and/or reduced interactivitywith antibodies to viral surface components including reducedsensitivity to these antibodies. The present invention furthercontemplates assays for detecting such viral variants, which assays areuseful in monitoring anti-viral therapeutic regimens and in developingnew or modified vaccines directed against viral agents and in particularthe resistant HBV variants of the present invention. The presentinvention also contemplates the use of the viral variants to screen foragents capable of inhibiting infection, replication and/or release ofthe virus.

2. Description of the Prior Art

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in any country.

Specific mutations in an amino acid sequence are represented herein as‘Xaa₁nXaa₂’ where Xaa₁ is the original amino acid residue beforemutation, n is the residue number and Xaa₂ is the mutant amino acid. Theabbreviation ‘Xaa’ may be the three letter or single letter (i.e. ‘X’)code. The amino acid residues for Hepatitis B virus DNA polymerase arenumbered with the residue methionine in the motif Tyr Met Asp Asp (YMDD)being residue number 204 (Stuyver et al., Hepatology 33: 751-757, 2001).The amino acid residues for hepatitis B virus surface antigen are numberaccording to Norder et al. (J. Gen. Virol. 74: 341-1348, 1993).

The term nucleoside analogs has been used in reference to bothnucleotide and nucleoside analogs.

Hepatitis B virus (HBV) can cause debilitating disease conditions andcan lead to acute liver failure. HBV is a DNA virus which replicates viaan RNA intermediate and utilizes reverse transcription in itsreplication strategy (Summers and Mason, Cell 29: 403-415, 1982). TheHBV genome is of a complex nature having a partially double-stranded DNAstructure with overlapping open reading frames encoding surface, core,polymerase and X genes. The complex nature of the HBV genome isrepresented in FIG. 1. The polymerase consists of four functionalregions, the terminal protein (TP), spacer, reverse transcriptase (rt)and ribonuclease (RNAse).

The polymerase gene of HBV overlaps the envelope gene, mutations in thecatalytic domain of the polymerase can affect the amino acid sequence ofthe envelope protein and vice versa. In particular, the genetic sequencefor the neutralization domain of HBV known as the ‘a’ determinant, whichis found within the HBsAg and located between amino acids 99 and 169,actually overlaps the major catalytic regions of the viral polymeraseprotein and in particular domains A and B.

The presence of an HBV DNA polymerase has led to the proposition thatnucleoside and nucleotide analogs could act as effective anti-viralagents. Examples of nucleoside analogs currently being tested arepenciclovir and its oral form (FAM or FCV) [Vere Hodge, Antiviral ChemChemother 4: 67-84, 1993; Boyd et al., Antiviral Chem Chemother. 32:358-363, 1987; Kruger et al., Hepatology 22: 219A, 1994; Main et al., J.Viral Hepatitis 3: 211-215, 1996] Lamivudine[(−)-β-2′-deoxy-3′-thiacytidine; (3TC or LMV) [Severini et al.,Antimicrobial Agents Chemother 39: 1430-1435, 1995; Dienstag et al., NewEngland J Med 333: 1657-1661, 1995]. New nucleoside analogs which havealready progressed to clinical trials include the pyriamidinesEmtricitabine, ((−)-β-L-2′-3′-dideoxy-5-fluoro-3′-thiacydidine; FTC),the 5-fluoro derivative of 3TC, and Clevudine(1-(2-fluoro-5-methyl-β-L-arabino-furanosyl) uracil; L-FMAU), athymidine analog. Like 3TC, these are pyrimidine derivatives with anunnatural “L”-configuration. Several purine derivatives have alsoprogressed to clinical trials; they include Entecavir (BMS-200,475;ETV), a carbocyclic deoxyguanosine analog, diaminopurine dioxolane(DAPD), an oral pro-drug for dioxolane guanine ((−)-β-D-2-aminopurinedioxolane; DXG) and Adefovir dipivoxil, an oral prodrug for the acyclicdeoxyadenosine monophosphate nucleoside analog Adefovir(9-[phosphonyl-methoxyethyl]-adenine; PMEA). Tenofovir disoproxilfumarate (TDF) has activity against HBV in vitro [Lada O et al., AntivirTher.; 9:353-63, 2004]. TDF has been approved for the treatment of HIVand has been used in co-infected HIV-HBV patients for the treatment ofboth HBV and HIV.

Whilst these agents are highly effective in inhibiting HBV DNAsynthesis, there is the potential for resistant mutants of HBV to emergeduring long term antiviral chemotherapy. In patients on prolonged LMVtherapy key resistance mutations are selected in the rt domain withinthe polymerase at rtM204I/V+/−rtL180M. The nomenclature used for thepolymerase mutations is in accordance with that proposed by Stuyver etal., 2001, supra. Only LMV and ADV have been approved for use againstchronic HBV infection. LMV and ADV are both potent inhibitors of HBVreplication and reduce HBV DNA viral load. Unfortunately, bothLMV-resistant and ADV-resistant strains of HBV are selected duringtherapy.

HIV and HBV are both blood borne viruses and have similar potentialroutes of transmission. Approximately 10% of HIV infected individualsare co-infected with HBV. The improved prognosis of HIV-infection thathas occurred since the introduction of highly active antiretroviraltherapy (HAART) has resulted in renewed emphasis being placed onco-morbidities associated with HIV-infection, and chronic viralhepatitis in particular. Hepatitis B virus (HBV) infection is animportant infection in HIV-1 infected individuals because of theinfluence of HIV-1 co-infection on the natural history of HBV infection.Antiviral therapies with activity against both viruses have enabledtargeted therapy in co-infected individuals. However, antiviralresistance is an important issue for both HIV and HBV.

TDF is also a potent inhibitor of HBV replication. TDF has activityagainst both hepadnaviruses and HIV. TDF was formerly referred to asbis-POC PMPA the oral derivative of9-[(R)-2-(phosphonomethoxy)propyl]adenine (PMPA) [Shaw J P et al., PharmRes. 14(12):1824-9, 1997]. Preclinical studies demonstrate that TDF is ahighly potent inhibitor of HBV in vitro and is effective against the LMVresistant viruses [Ying C et al., J Viral Hepat. 7(2):161-5, 2000]. TDFhas been used successfully to treat patients with the LMV resistant HBVmutations. No specific TDF resistant mutations had been described forHBV.

Nucleoside or nucleotide analog therapy may be administered asmonotherapy or combination therapy where two or more nucleoside ornucleotide analogs may be administered. There is a need to monitor fordevelopment of nucleoside-/nucleotide-resistant variants of HBV as wellas variants resistant to other HBV DNA polymerase antagonists andvariants resistant to antibodies to HBV surface components. The rapididentification can lead to altered therapeutic protocols being pursued.In addition, resistant HBV variants are useful as targets for screeningof new antiviral agents.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1, <400>2, etc. A sequence listing isprovided after the claims.

The positions of nucleotide and amino acid mutations identified usingnomenclature from genotypes B, C or F where the methionine residue inthe YMDD motif of the DNA polymerase was designated position 550 (seeAustralian Patent No. 734831). The nucleotide and amino acid positionsgiven in the present specification are based on a new nomenclature wherethe methionine residue is YMDD is position 204 and is referred to asrtM204 where rt is an abbreviation for “reverse transcriptase”.

In accordance with the present invention, HBV resistant variants wereidentified in two patients (patient A and patient B) with chronichepatitis B and HIV, treated with both LMV and TDF. In combinationtherapy, accordance with the present invention, resistant variants ofHBV were identified, following LMV and TDF treatment, with mutations inthe HBV DNA polymerase gene which reduce the sensitivity of HBV tonucleoside and nucleotide analogs. The identification of these HBVvariants is important for the development of assays to monitor LMVand/or TDF resistance and/or resistance to other nucleoside ornucleotide analog therapeutic regimes and to screen for agents which areuseful as alternative therapeutic agents. The mutations detected in theHBV isolated from patients A and B were the LMV resistant mutationsnamely the rtV173L+rtL180M+rtM204V in association with a unique mutationat rtA194T.

The detection of such HBV variants is particularly important in themanagement of therapeutic protocols including the selection ofappropriate agents for treating HBV infection. The method of this aspectof the present invention is predicated in part on monitoring thedevelopment in a subject of an increased HBV load in the presence of anucleoside or nucleotide analog and/or the presence of antibodies toviral surface components. The clinician is then able to modify anexisting treatment protocol or select an alternative treatment protocolaccordingly. In addition, the present invention encompasses HBV variantsobtained from subjects following exposure to LMV and/or TDF but whichare still sensitive to one or both of LMV and/or TDF. Such variants mayexhibit resistance to other nucleoside or nucleotide analogs and/or toantibodies to viral surface components.

One aspect of the present invention, therefore, is directed to anisolated HBV variant comprising a nucleotide mutation in a gene encodinga DNA polymerase resulting in at least one amino acid addition,substitution and/or deletion to the DNA polymerase and which exhibitsdecreased sensitivity to TDF and/or LMV and optionally, in addition oralternatively, other nucleoside or nucleotide analogs, other HBV DNApolymerase antagonists and/or or immunological reagents. In this regard,exposure of a patient to TDF and/or LMV may result in the selection ofan HBV variant which exhibits resistance or reduced sensitivity to anucleoside or nucleotide analog other than TDF or LMV or to animmunology reagent such as an antibody to an HBV surface antigen.Preferably, the DNA polymerase exhibits reduced sensitivity to TDF, orand LMV. The variant HBV comprises a mutation in an overlapping openreading frame in its genome in a region defined by one or more ofdomains F and A through E of HBV DNA polymerase.

The present invention further contemplates a method for determining thepotential for an HBV to exhibit reduced sensitivity to TDF and/or LMV oroptionally, in addition or alternatively, other nucleoside or nucleotideanalogs, other HBV DNA polymerase antagonists and/or immunologicalreagents by isolating DNA or corresponding mRNA from the HBV andscreening for a mutation in the nucleotide sequence encoding HBV DNApolymerase resulting in at least one amino acid substitution, deletionand/or addition in any one or more of domains F and A through E or aregion proximal thereto of the DNA polymerase and associated withresistance or decreased sensitivity to TDF and/or LMV. The presence ofsuch a mutation is an indication of the likelihood of resistance to saidentecavir and/or LMV. Preferably, the HBV variant exhibits reducedsensitivity to TDF, or both TDF and LMV.

The present invention also provides a composition comprising a variantHBV resistant to TDF and/or LMV and optionally, in addition oralternatively, other nucleoside or nucleotide analogs, other HBV DNApolymerase antagonists and/or immunological reagents or a recombinant orderivative form thereof or its chemical equivalent and one or morepharmaceutically acceptable carriers and/or diluents. Yet another aspectof the present invention provides a use of the aforementionedcomposition or a variant HBV comprising a nucleotide mutation in a geneencoding a DNA polymerase resulting in at least one amino acid addition,substitution and/or deletion to the DNA polymerase and a decreasedsensitivity to TDF and/or LMV and optionally in addition oralternatively other nucleoside or nucleotide analogs or other HBV DNApolymerase antagonists or immunological reagents in the manufacture of amedicament for the treatment and/or prophylaxis of HBV infection.

The present invention also contemplates a method for determining whetheran HBV strain exhibits reduced sensitivity to a nucleoside or nucleotideanalog, or other HBV DNA polymerase antagonist by isolating DNA orcorresponding mRNA from the HBV and screening for a mutation in thenucleotide sequence encoding the DNA polymerase wherein the presence ofthe following mutations in the rt region: rtV173L, rtL180M, rtA194T andrtM204V or combinations thereof or an equivalent one or more othermutation is indicative of a variant which exhibits a decreasedsensitivity to TDF and/or LMV and optionally other nucleoside ornucleotide analogs and/or other HBV DNA polymerase antagonists and/or animmunological reagent.

The subject method may also be practiced by screening for a mutation inthe nucleotide sequence encoding the DNA polymerase wherein the presenceof the following mutations in the B or C domain of the rt region:rtV173L, rtL180M, rtA194T and rt M204V or combinations thereof or anequivalent one or more other mutations is indicative of a variant whichexhibits a decreased sensitivity to TDF and/or LMV and optionally othernucleoside or nucleotide analogs, or other HBV DNA polymeraseantagonists and/or an immunological reagent.

It should be noted that mutants, rtV173L, rtL180M and rtM204V correspondto mutants, V519L, L526M, and M550V, respectively in Australian PatentNo. 734831 (using an earlier nomenclature system).

Still a further methodology comprises screening for a mutation in thenucleotide sequence encoding the envelope genes wherein the presence ofthe following mutations in the PreS1, PreS2 and S genes (changes in theoverlapping reverse transcriptase region are indicated in parenthesis):sE164D (=rtV173L), sS193L (no change), sI195M (=rtM204V), combinationsthereof or an equivalent one or more other mutation is indicative of avariant which exhibits a decreased sensitivity to TDF and/or LMV andoptionally other nucleoside or nucleotide analogs, or other HBV DNApolymerase antagonists and/or an immunological reagent.

Another aspect of the present invention is directed to a variant HBVcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion or atruncation compared to a surface antigen from a reference or wild typeHBV and wherein an antibody generated to the reference or wild typesurface antigen exhibits an altered immunological profile relative tosaid HBV variant. One altered profile includes a reduced capacity forneutralizing the HBV. More particularly, the surface antigen of thevariant HBV exhibits an altered immunological profile compared to apre-treatment HBV where the variant HBV is selected for by a nucleosideor nucleotide analog or other antagonist of the HBV DNA polymerase. Thevariant HBV of this aspect of the invention may also comprise anucleotide sequence comprising a single or multiple nucleotidesubstitution, addition and/or deletion compared to a pre-treatment HBV.

The present invention extends to an isolated HBsAg or a recombinant formthereof or derivative or chemical equivalent thereof corresponding tothe variant HBV. Generally, the HBsAg or its recombinant or derivativeform or its chemical equivalent comprises an amino acid sequence with asingle or multiple amino acid substitution, addition and/or deletion ora truncation compared to an HBsAg from a reference HBV and wherein anantibody directed to a reference HBV exhibits an altered immunologicalprofile to an HBV carrying said variant HBsAg. In one embodiment, thealtered immunological profile comprises a reduction in the ability toneutralize the variant HBV.

The present invention is predicated in part on the identification andisolation of variants of HBV that have a plurality of mutations andexhibit two or more characteristics selected from decreased or reducedsensitivity to one or more nucleoside or nucleotide analogs or other HBVDNA polymerase antagonist, a reduced level and/or functional activity ofHepatitis Be antigen, or a reduced, abrogated or otherwise impairedimmunological interactivity, relative to wild-type HBV. Thus, theidentification of HBV variants with these mutational patterns isimportant inter alia for the development of assays to detect HBVvariants and assays to screen for agents which are useful in treatingand/or preventing infections by those variants and/or other HBV isolatesand for the development of alternative therapeutic regimes for managingHBV infections.

Accordingly, one aspect of the present invention is directed to anisolated HBV variant comprising a plurality of nucleotide mutations thatcorrelate with at least two characteristics selected from (a) resistanceto one or more nucleoside analogs, (b) a reduced level and/or functionalactivity of Hepatitis Be antigen, or (c) a reduced, abrogated orotherwise impaired immunological interactivity.

Another aspect of the present invention contemplates an isolated HBVvariant comprising a plurality of nucleotide mutations that correlatewith (a) resistance to one or more nucleoside or nucleotide analogs orother antagonists of HBV DNA polymerase activity, (b) a reduced leveland/or functional activity of Hepatitis Be antigen, and (c) a reduced,abrogated or otherwise impaired immunological interactivity.

Yet another aspect of the present invention provides an isolated HBVvariant comprising a plurality of nucleotide mutations selected from twoor more of (a) a nucleotide mutation in a gene encoding a DNA polymeraseresulting in at least one amino acid addition, substitution and/ordeletion to said DNA polymerase wherein said variant exhibits decreasedsensitivity to TDF and/or LMV and optionally other nucleoside ornucleotide analogs or other antagonists of HBV DNA polymerase activity,(b) a nucleotide mutation in a gene encoding a Hepatitis Be antigen orin a transcriptional control element of said gene wherein said mutationresults in a reduced level and/or functional activity of said HepatitisBe antigen, or (c) a nucleotide mutation in a gene encoding a hepatitisB polypeptide resulting in at least one amino acid addition,substitution and/or deletion to said polypeptide which reduces,abrogates or otherwise impairs its immunological interactivity.

Another aspect of the present invention contemplates a method fordetecting an agent which exhibits inhibitory activity to an HBV bygenerating a genetic construct comprising a replicationcompetent-effective amount of the genome from the HBV contained in aplasmid vector and then transfecting said cells with said construct,contacting the cells, before, during and/or after transfection, with theagent to be tested, culturing the cells for a time and under conditionssufficient for the HBV to replicate, express genetic sequences and/orassemble and/or release virus or virus-like particles if resistant tosaid agents; and the subjecting the cells, cell lysates or culturesupernatant fluid to viral- or viral-component-detection means todetermine whether or not the virus has replicated, expressed geneticmaterial and/or assembled and/or been released in the presence of theagent. In a preferred embodiment, the plasmid vector in a baculovirusvector and the method comprises generating a genetic constructcomprising a replication competent-effective amount of the genome fromthe HBV contained in or fused to an amount of a baculovirus genomeeffective to infect cells and then infecting said cells with saidconstruct, contacting the cells, before, during and/or after infection,with the agent to be tested, culturing the cells for a time and underconditions sufficient for the HBV to replicate, express geneticsequences and/or assemble and/or release virus or virus-like particlesif resistant to said agent and then subjecting the cells, cell lysatesor culture supernatant fluid to viral- or viral-component-detectionmeans to determine whether or not the virus has replicated, expressedgenetic material and/or assembled and/or been released in the presenceof the agent.

In an alternative embodiment, the method comprises generating acontinuous cell line comprising an infectious copy of the genome of theHBV in a replication competent effective amount such that saidinfectious HBV genome is stably integrated into said continuous cellline such as but not limited to 2.2.15 or AD, contacting the cells withthe agent to be tested, culturing the cells for a time and underconditions sufficient for the HBV to replicate, express geneticsequences and/or assemble and/or release virus or virus-like particlesif resistant to the agent and then subjecting the cells, cell lysates orculture supernatant fluid to viral- or viral-component-detection meansto determine whether or not the virus has replicated, expressed geneticmaterial and/or assembled and/or been released in the presence of theagent.

In an alternative embodiment, the present invention also contemplate amethod for detecting an agent which exhibits inhibitory activity to anHBV polymerase in an in vitro polymerase assay. The HBV polymeraseactivity can be examined using established assays (Gaillard et al.,Antimicrob Agents Chemother. 46(4): 1005-1013, 2002; Xiong et al.,Hepatology. 28(6): 1669-73, 1998). The HBV polymerase may be a wild-typeor reference HBV polymerase or mutant HBV polymerase.

In connection with these methods, the plasmid vector may include genesencoding part or all of other viral vectors such as baculovirus vectorsor adenovirus vectors (see Ren and Nassal, J. Virol. 75(3): 1104-1116,2001).

The identification of viral variants enables the production of vaccinescomprising particular recombinant viral components such as polymerasesor envelope genes PreS1, PreS2, S encoding for L, M, S proteins as wellas therapeutic vaccines comprising defective HBV variants. Rational drugdesign may also be employed to identify or generate therapeuticmolecules capable of interacting with a polymerase or envelope genesPreS1, PreS2, S encoding for L, M, S proteins or other component of theHBV. Such drugs may also have diagnostic potential.

The abbreviations defined in Table 1 are used in the subjectspecification

TABLE 1 Abbreviations ABBREVIATION DESCRIPTION 3TC (LMV);(−)-β-2′-deoxy-3′-thiacytidine ADV Adefovir DAPD diaminopurine dioxolaneDDI Didanosine DXG dioxolane guanine ETV Entecavir FAM Famciclovir FTCEmtricitabine HBsAg hepatitis B surface antigen HBV hepatitis B virusHIV human immunodeficiency virus LMV Lamividuine LPV/rLopinavir/ritonavir nt nucleotide PMEA Adefovir PMPA9-[(R)-2-(phosphonomethoxy)propyl]adenine RNAse Ribonuclease rt reversetranscriptase TDF Tenofovir disoproxil fumarate YMDD Tyr Met Asp Asp-amotif in the polymerase protein; where the Met residue is designatedresidue number 204 of the reverse transcriptase

A summary of sequence identifiers used throughout the subjectspecification is provided in Table 2.

TABLE 2 Summary of sequence identifiers SEQUENCE ID NO: DESCRIPTION 1primer 1 forward 2 primer 2 forward 3 primer 3 reverse 4 primer 4reverse 5 Patient A HBV nucleotide (nt) sequence encoding catalyticregion of DNA polymerase Week 0 (FIG. 3) 6 Patient A HBV amino acidsequence of catalytic region of DNA polymerase Week 0 (FIG. 4) 7 PatientA HBV amino acid sequence of surface antigen (HBsAg) Week 0 (FIG. 5) 8Patient A HBV nt sequence of encoding catalytic region of DNA polymeraseWeek 48 (FIG. 6) 9 Patient A HBV amino acid sequence of catalytic regionof DNA polymerase gene Week 48 (FIG. 7) 10 Patient A HBV amino acidsequence of surface antigen (HBsAg) Week 48 (FIG. 8) 11 Patient A HBV ntsequence of encoding catalytic region of DNA polymerase Week 62 (FIG. 9)12 Patient A HBV amino acid sequence of catalytic region of DNApolymerase gene Week 62 (FIG. 10) 13 Patient A HBV amino acid sequenceof surface antigen (HBsAg) Week 62 (FIG. 11) 14 Patient B HBV ntsequence of encoding catalytic region of DNA polymerase Week 77 (FIG.12) 15 Patient B HBV amino acid sequence of catalytic region of DNApolymerase gene Week 77 (FIG. 13) 16 Patient B HBV amino acid sequenceof surface antigen (HbsAg) Week 77 (FIG. 14) 17 rtM204V forward primer18 rtM204V reverse primer 19 rt180 forward primer 20 rt180 reverseprimer 21 A194T forward primer 22 A194T reverse primer 23 3FL-XHybprobeprobe 24 5LC-XHybprobe probe 25 X sense probe 26 X antisense probe

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatic representation showing the partially doublestranded DNA HBV genome showing the overlapping open reading framesencoding surface (S), core (C), polymerase (P) and X gene.

FIG. 2 is a diagrammatic representation of a computer system fordetermining the potency value (P_(A)) of a variant HBV.

FIG. 3 is a representation showing the HBV nucleotide sequence encodingthe catalytic region of the polymerase gene from Patient A at week 0 ofTDF treatment.

FIG. 4 is a representation showing the deduced amino acid sequence ofthe catalytic region of the polymerase gene from Patient A at week 0 ofTDF treatment.

FIG. 5 is a representation showing the deduced amino acid sequence ofthe envelope gene from Patient A week 0 of TDF treatment.

FIG. 6 is a representation showing the HBV nucleotide sequence encodingthe catalytic region of the polymerase gene from Patient A at week 48 ofTDF treatment.

FIG. 7 is a representation showing the deduced amino acid sequence ofthe catalytic region of the polymerase gene from Patient A at week 48 ofTDF treatment.

FIG. 8 is a representation showing the deduced amino acid sequence ofthe envelope gene from Patient A week 48 of TDF treatment.

FIG. 9 is a representation showing the HBV nucleotide sequence encodingthe catalytic region of the polymerase gene from Patient A at week 62 ofTDF treatment.

FIG. 10 is a representation showing the deduced amino acid sequence ofthe catalytic region of the polymerase gene from Patient A at week 62 ofTDF treatment.

FIG. 11 is a representation showing the deduced amino acid sequence ofthe envelope gene from Patient A week 62 of TDF treatment.

FIG. 12 is a representation showing the HBV nucleotide sequence encodingthe catalytic region of the polymerase gene from Patient B at week 77 ofTDF treatment.

FIG. 13 is a representation showing the deduced amino acid sequence ofthe catalytic region of the polymerase gene from Patient B at week 77 ofTDF treatment.

FIG. 14 is a representation showing the deduced amino acid sequence ofthe envelope gene from Patient B week 77 of TDF treatment.

FIG. 15 is a diagrammatic representation of the HBV polymerase. Residueaa194 from the wild type is highlighted in FIG. 15 b. Residue aa194T ishighlighted in FIG. 15 c.

FIG. 16 is a diagrammatic representation of the chemical structure ofTDF.

FIG. 17 is a graphical representation showing HBV DNA version TDFtreatment in (a) Patient A and (b) Patient B.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention is predicated in part on the identification andisolation of nucleoside or nucleotide analog resistant variants of HBVor variants resistant to other DNA polymerase antagonists orimmunological reagents which bind to a surface compound of HBV followingtreatment of patients with TDF or LMV or TDF and LMV and optionallyother nucleoside or nucleotide analogs. In particular, TDF, or TDF andLMV treated patients gave rise to variants of HBV exhibiting decreasedor reduced sensitivity to TDF and/or LMV. Reference herein to“decreased” or “reduced” in relation to sensitivity to TDF and/or LMVincludes and encompasses a complete or substantial resistance to thenucleoside or nucleotide analog as well as partial resistance andincludes a replication rate or replication efficiency (yield phenotype)which is more than a wild-type in the presence of a nucleoside ornucleotide analog or other DNA polymerase antagonist. In one aspect,this is conveniently measured by an increase in viral load to a levelsimilar or greater than pre-treatment levels.

Accordingly, one aspect of the present invention is directed to anisolated HBV variant wherein said variant comprises a nucleotidemutation in a gene encoding a DNA polymerase resulting in at least oneamino acid addition, substitution and/or deletion to said DNA polymeraseand wherein said variant exhibits decreased sensitivity to TDF and/orLMV and optionally other nucleoside or nucleotide analogs, or other HBVDNA polymerase antagonists and/or an immunological reagent.

In a related aspect, the present invention provides an isolatedHepatitis B virus (HBV) variant wherein said variant comprises anucleotide mutation in a gene encoding a DNA polymerase resulting in atleast one amino acid addition, substitution and/or deletion to said DNApolymerase and wherein said variant either exhibits decreasedsensitivity to one or more of TDF and/or LMV or is selected followingexposure to one or both of TDF and/or LMV.

Preferably, the decreased sensitivity is in respect of TDF, or both TDFand LMV. Alternatively or in addition, the decreased sensitivity is inrespect of a nucleoside or nucleotide analog other than TDF or LMVand/or may be in respective of an immunological reagent such as anantibody to a surface compound of HBV.

In addition to a mutation in the gene encoding DNA polymerase, due tothe overlapping nature of the HBV genome (FIG. 1), a correspondingmutation may also occur in the gene encoding the surface antigen (HBsAg)resulting in reduced interactivity of immunological reagents such asantibodies and immune cells to HBsAg. The reduction in the interactivityof immunological reagents to a viral surface component generallyincludes the absence of immunological memory to recognise orsubstantially recognize the viral surface component. The presentinvention extends, therefore, to an HBV variant exhibiting decreasedsensitivity to TDF and/or LMV and reduced interactivity of animmunological reagent to HBsAg wherein the variant is selected forfollowing TDF and/or LMV combination or sequential treatment. The term“sequential” in this respect means TDF followed by LMV or LMV followedby TDF or multiple sequential administrations of each of TDF and LMV orLMV and TDF.

A viral variant may, therefore, carry mutation only in the DNApolymerase or both in the DNA polymerase and the HBsAg. The term“mutation” is to be read in its broadest context and includes multiplemutations.

The present invention extends to a mutation and any domain of the HBVDNA polymerase and in particular regions F and A through E provided saidmutation leads to decreased sensitivity to LMV and/or TDF.

Preferably, the mutation results in an altered amino acid sequence inany one or more of domains F and A through E or regions proximal theretoof the HBV DNA polymerase.

Another aspect of the present invention provides an HBV variantcomprising a mutation in an overlapping open reading frame in its genomewherein said mutation is in a region defined by one or more of domains Fand A through E of HBV DNA polymerase and wherein said variant exhibitsdecreased sensitivity to TDF and/or LMV and optionally other nucleosideor nucleotide analogs, or other HBV DNA polymerase antagonists and/or animmunological reagent.

In a related embodiment, there is provided an HBV variant comprising amutation in the nucleotide sequence encoding a DNA polymerase resultingin an amino acid addition, substitution and/or deletion in said DNApolymerase in one or more amino acids and wherein said variant exhibitsdecreased sensitivity to TDF and/or LTV and optionally other nucleosideor nucleotide analogs, or other HBV DNA polymerase antagonists and/or animmunological reagent. Preferably, the decreased sensitivity is to TDF,or both LMV and/or TDF.

The term “combination therapy” means that both TDF and LMV areco-administered in the same composition or simultaneously in separatecompositions. The term “sequential therapy” means that the two agentsare administered within seconds, minutes, hours, days or weeks of eachother and in either order. Sequential therapy also encompassescompleting a therapeutic course with one or other of TDF or LMV and thencompleting a second therapeutic course with the other of TDF or LMV.

Accordingly, another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said the variant HBV isselected for by a nucleoside or nucleotide analog of the HBV DNApolymerase or other antagonist of the DNA polymerase, said variantselected by exposure of a subject to TDF and/or LMV in combination orsequential therapy.

In a related embodiment, the present invention provides an HBV variantcomprising a nucleotide sequence comprising a single or multiplenucleotide substitution, addition and/or deletion compared to thepretreatment HBV and which HBV variant has a surface antigen exhibitingan altered immunological profile compared to the pretreatment HBV, saidvariant selected by exposure of a subject to TDF and/or LMV incombination or sequential therapy.

Preferably, the variants are in isolated form such that they haveundergone at least one purification step away from naturally occurringbody fluid. Alternatively, the variants may be maintained in isolatedbody fluid or may be in DNA form. The present invention alsocontemplates infectious molecular clones comprising the genome or partsthereof from a variant HBV. Furthermore, the present invention providesisolated components from the variant HBVs such as but not limited to anisolated HBsAg. Accordingly, the present invention provides an isolatedHBsAg or a recombinant form thereof or derivative or chemical equivalentthereof, said HBsAg being from a variant HBV selected by exposure of asubject to TDF and/or LMV in combination or sequential therapy.

More particularly, yet another aspect of the present invention isdirected to an isolated variant HBsAg or a recombinant or derivativeform thereof or a chemical equivalent thereof wherein said HBsAg or itsrecombinant or derivative form or its chemical equivalent exhibits analtered immunological profile compared to an HBsAg from a reference HBV,said HBsAg being from a variant HBV selected by exposure of a subject toTDF and/or LMV in combination or sequential therapy.

Even more particularly, the present invention provides an isolatedvariant HBsAg or a recombinant or derivative form thereof or a chemicalequivalent thereof wherein said HBsAg or its recombinant or derivativeform or its chemical equivalent comprises an amino acid sequence with asingle or multiple amino acid substitution, addition and/or deletion ora truncation compared to an HBsAg from a reference HBV and wherein aneutralising antibody directed to a reference HBV exhibits no or reducedneutralising activity to an HBV carrying said variant HBsAg, said HBsAgbeing from a variant HBV selected by exposure of a subject to TDF and/orLMV in combination or sequential therapy.

Preferred mutations in the HBV DNA polymerase include variants selectedfrom patients with HBV recurrence following TDF and/or LMV treatment.Preferably, the treatment involves TDF or both TDF and/or LMV incombination or sequential therapy. Nucleoside or nucleotide analogtreatment or other DNA polymerase antagonist treatment may occur inrelation to the treatment of patients diagnosed with hepatitis inpatients with HBV alone or with HBV/HIV co-infection. Followingselection of variants, viral loads are obtainable at levels greater thanpre-treatment levels.

Preferred mutations in the HBV DNA polymerase include one or more ofrtV173L, rtL180M, rtA194T, rtM204V, or combinations thereof or anequivalent one or more other mutation is indicative of a variant whereinsaid variant exhibits a decreased sensitivity to TDF and/or LMV andoptionally other nucleoside or nucleotide analogs, or other HBV DNApolymerase antagonists and/or an immunological reagent. It should benoted that the nomenclature system for amino acid positions is based onthe methionine residues in the YMDD motif being designated codon rtM204.This numbering system is different to that in Australian Patent No.734831 where the methionine residue in the YMDD motif within thepolymerase gene is designated codon 550. In this regard, rtV173L,rtL180M and rtM204V correspond to V519L, L526M and M550V, respectively,in Australian Patent No. 734831. Corresponding mutations may also occurin envelope genes such as in one or more of PreS1, PreS2 and HBsAg.Particular mutations are as follows: sE164D, sS193L, sI195M, orcombinations thereof or an equivalent one or more other mutation isindicative of a variant wherein said variant exhibits a decreasedsensitivity to TDF and/or LMV and optionally other nucleoside ornucleotide analogs, or other HBV DNA polymerase antagonists and/or animmunological reagent. The mutations in gene encoding HBsAg at sE164D,or sI195M also result in mutation in the in the polymerase gene rtV173L,or rtM204V respectively.

The identification of the variants of the present invention permits thegeneration of a range of assays to detect such variants. The detectionof such variants may be important in identifying resistant variants todetermine the appropriate form of chemotherapy.

Still another aspect of the present invention contemplates a method fordetermining the potential for an HBV to exhibit reduced sensitivity toTDF and/or LMV or optionally other nucleoside or nucleotide analogs, orother HBV DNA polymerase antagonists and/or an immunological reagent,said method comprising isolating DNA or corresponding mRNA from said HBVand screening for a mutation in the nucleotide sequence encoding HBV DNApolymerase resulting in at least one amino acid substitution, deletionand/or addition in any one or more of domains F and A through E or aregion proximal thereto of said DNA polymerase and associated withresistance or decreased sensitivity to TDF and/or LMV wherein thepresence of such a mutation is an indication of the likelihood ofresistance to said TDF and/or LMV.

Preferably, the assay detects one or more of the following mutations inthe rt region: rtV173L, rtL180M, rtA194T, rtM204V, or combinationsthereof or an equivalent one or more other mutation is indicative of avariant wherein said variant exhibits a decreased sensitivity to TDFand/or LMV and optionally other nucleoside or nucleotide analogs, orother HBV DNA polymerase antagonists and/or an immunological reagent.

Accordingly, another aspect of the present invention produces a methodfor determining whether an HBV strain exhibits reduced sensitivity to anucleoside or nucleotide analogs, or other HBV DNA polymeraseantagonists and/or an immunological reagent, said method comprisingisolating DNA or corresponding mRNA from said HBV and screening for amutation in the nucleotide sequence encoding the DNA polymerase whereinthe presence of the following mutations in the rt region: rtV173L,rtL180M, rtA194T, rtM204V, or combinations thereof or an equivalent oneor more other mutation is indicative of a variant wherein said variantexhibits a decreased sensitivity to TDF and/or LMV and optionally othernucleoside or nucleotide analogs.

In a related embodiment, the present invention contemplates a method fora method for determining the potential for an HBV to exhibit reducedsensitivity to a nucleoside or nucleotide analog selected from TDF andLMV or optionally other nucleoside or nucleotide analogs or animmunological agent, said method comprising isolating DNA orcorresponding mRNA from said HBV and screening for a mutation in thenucleotide sequence encoding HBV DNA polymerase resulting in at leastone amino acid substitution, deletion and/or addition in any one or moreof domains F and A through E or a region proximal thereto of said DNApolymerase and associated with resistance or decreases sensitivity toone or more of TDF and/or LMV and/or an immunological agent wherein thepresence of such a mutation is an indication of the likelihood ofresistance to said one or more of TDF, LMV or an immunological reagent.

The preferred mutation in the reverse transcriptase is at rtA194T orcombinations thereof or an equivalent one or more other mutation isindicative of a variant wherein said variant exhibits a decreasedsensitivity to TDF and/or LMV and optionally other nucleoside ornucleotide analogs, or other HBV DNA polymerase antagonists and/or animmunological reagent.

Accordingly, another aspect of the present invention contemplates amethod for determining whether an HBV strain exhibits reducedsensitivity to a nucleoside or nucleotide analogs, or other HBV DNApolymerase antagonists and/or an immunological reagent, said methodcomprising isolating DNA or corresponding mRNA from said HBV andscreening for a mutation in the nucleotide sequence encoding the DNApolymerase wherein the presence of the following mutations in the B or Cdomain, or the B/C interdomain of the rt region:, rtV173L, rtL180M,rtA194T, rtM204V or combinations thereof or an equivalent one or moreother mutation is indicative of a variant wherein said variant exhibitsa decreased sensitivity to TDF and/or LMV and optionally othernucleoside or nucleotide analogs, or other HBV DNA polymeraseantagonists and/or an immunological reagent.

The detection of HBV or its components in cells, cell lysates, culturedsupernatant fluid and bodily fluid may be by any convenient meansincluding any nucleic acid-based detection means, for example, bynucleic acid hybridization techniques or via one or more polymerasechain reactions (PCRs). The term “bodily fluid” includes any fluidderived from the blood, lymph, tissue or organ systems including serum,whole blood, biopsy and biopsy fluid, organ explants and organsuspension such as liver suspensions. The invention further encompassesthe use of different assay formats of said nucleic acid-based detectionmeans, including restriction fragment length polymorphism (RFLP),amplified fragment length polymorphism (AFLP), single-strand chainpolymorphism (SSCP), amplification and mismatch detection (AMD),interspersed repetitive sequence polymerase chain reaction (IRS-PCR),inverse polymerase chain reaction (iPCR) and reverse transcriptionpolymerase chain reaction (RT-PCR), amongst others. Other forms ofdetection include Northern blots, Southern blots, PCR sequencing,antibody procedures such as ELISA, Western blot andimmunohistochemistry. A particularly useful assay includes the reagentsand components required for immobilized oligonucleotide- oroligopeptide-mediated detection systems.

One particularly useful nucleic acid detection system is the reversehybridization technique. In this technique, DNA from an HBV sample isamplified using a biotin or other ligand-labeled primer to generate alabeled amplificon. Oligonucleotides immobilized to a solid support suchas a nitrocellulose film are then used to capture amplified DNA byhybridization. Specific nucleic acid fragments are identified via biotinor the ligand. Generally, the labeled primer is specific for aparticular nucleotide variation to be detected. Amplification occursonly if the variation to be detected is present. There are many forms ofthe reverse hybridization assay and all are encompassed by the presentinvention.

Detecting HBV replication in cell culture is particularly useful.

Another aspect of the present invention contemplates a method fordetecting an agent which exhibits inhibitory activity to an HBV by:

-   -   generating a genetic construct comprising a replication        competent-effective amount of the genome from the HBV contained        in a plasmid vector and then transfecting said cells with said        construct;    -   contacting the cells, before, during and/or after transfection,        with the agent to be tested;    -   culturing the cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to said agents; and    -   then subjecting the cells, cell lysates or culture supernatant        fluid to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of the agent.

In a preferred embodiment, the plasmid vector may include genes encodingpart or all of other viral vectors such as baculovirus or adenovirus(Ren and Nassal, 2001, supra) and the method comprises:

-   -   generating a genetic construct comprising a replication        competent-effective amount of the genome from the HBV contained        in or fused to an amount of a baculovirus genome or adenovirus        genome effective to infect cells and then infecting said cells        with said construct;    -   contacting the cells, before, during and/or after infection,        with the agent to be tested;    -   culturing the cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to said agent; and    -   then subjecting the cells, cell lysates or culture supernatant        fluid to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of the agent.

In an alternative embodiment, the method comprises:

-   -   generating a continuous cell line comprising an infectious copy        of the genome of the HBV in a replication competent effective        amount such that said infectious HBV genome is stably integrated        into said continuous cell line such as but not limited to 2.2.15        or AD;    -   contacting the cells with the agent to be tested;    -   culturing the cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to the agent; and    -   then subjecting the cells, cell lysates or culture supernatant        fluid to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of the agent.

As indicated above, variants may also be detected with reference to theHBsAg (s gene) and Pres1, Pres2 envelop genes. Preferred mutations inthis regard include one or more of sE164D, sS193L, or sI195M. The abovemethods are useful for detecting an agent which exhibits inhibitoryactivity to an HBV which exhibits resistance to one or more of TDF, LMV,a nucleoside analog other than TDF or LMV or an immunological reagent.

Accordingly, another aspect of the present invention contemplates amethod for determining whether an HBV strain exhibits reducedsensitivity to a nucleoside or nucleotide analogs, or other HBV DNApolymerase antagonists and/or an immunological reagent, said methodcomprising isolating DNA or corresponding mRNA from said HBV andscreening for a mutation in the nucleotide sequence encoding theenvelope genes wherein the presence of the following mutations in theHBsAg: sE164D, sS193L, or sI195M or combinations thereof or anequivalent one or more other mutation is indicative of a variant whereinsaid variant exhibits a decreased sensitivity to TDF and/or LMV andoptionally other nucleoside or nucleotide analogs, or other HBV DNApolymerase antagonists and/or an immunological reagent.

The present invention is predicated in part on the identification andisolation of variants of HBV that have a plurality of mutations andexhibit two or more characteristics selected from decreased or reducedsensitivity to one or more nucleoside or nucleotide analogs or otherantagonists of HBV DNA polymerase activity, a reduced level and/orfunctional activity of Hepatitis Be antigen, or a reduced, abrogated orotherwise impaired immunological interactivity, relative to wild-typeHBV. Thus, the identification of HBV variants with these mutationalpatterns is important inter alia for the development of assays to detectHBV variants and assays to screen for agents which are useful intreating and/or preventing infections by those variants and/or other HBVisolates and for the development of alternative therapeutic regimes formanaging HBV infections.

Accordingly, one aspect of the present invention is directed to anisolated HBV variant comprising a plurality of nucleotide mutations thatcorrelate with at least two characteristics selected from (a) resistanceto one or more nucleoside or nucleotide analogs or other antagonists ofHBV DNA polymerase activity, (b) a reduced level and/or functionalactivity of Hepatitis Be antigen, or (c) a reduced, abrogated orotherwise impaired immunological interactivity.

Another aspect of the present invention contemplates an isolated HBVvariant comprising a plurality of nucleotide mutations that correlatewith (a) resistance to one or more nucleoside or nucleotide analogs orother antagonists of HBV DNA polymerase activity, (b) a reduced leveland/or functional activity of Hepatitis Be antigen, and (c) a reduced,abrogated or otherwise impaired immunological interactivity.

Yet another aspect of the present invention provides an isolated HBVvariant comprising a plurality of nucleotide mutations selected from twoor more of (a) a nucleotide mutation in a gene encoding a DNA polymeraseresulting in at least one amino acid addition, substitution and/ordeletion to said DNA polymerase wherein said variant exhibits decreasedsensitivity to TDF and/or LMV and optionally other nucleoside ornucleotide analogs or other antagonists of HBV DNA polymerase activity,(b) a nucleotide mutation in a gene encoding a Hepatitis Be antigen orin a transcriptional control element of said gene wherein said mutationresults in a reduced level and/or functional activity of said HepatitisBe antigen, or (c) a nucleotide mutation in a gene encoding a hepatitisB polypeptide resulting in at least one amino acid addition,substitution and/or deletion to said polypeptide which reduces,abrogates or otherwise impairs its immunological interactivity.

The detection of amino acid variants of DNA polymerase is convenientlyaccomplished by comparison to reference sequences. The polymorphismsshown represent the variations shown in various databases for activepathogenic HBV strains. Where an HBV variant comprises an amino aciddifferent to what is represented, then such an isolate is considered aputative HBV variant having an altered DNA polymerase activity.

The present invention further contemplates agents which inhibit TDFand/or LMV resistant HBV variants. Such agents will be particularlyuseful if long-term treatment by TDF and/or LMV and/or optionally othernucleoside or nucleotide analogs, or other HBV DNA polymeraseantagonists and/or an immunological reagent is contemplated by theclinician. The agents may be DNA or RNA or proteinaceous ornon-proteinaceous chemical molecules. Natural product screening such asfrom plants, coral and microorganisms is also contemplated as a usefulpotential source of masking agents. The agents may be in isolated formor in the form of a pharmaceutical composition and may be administeredsequentially or simultaneously with the nucleoside or nucleotideanalogs, or other HBV DNA polymerase antagonists and/or an immunologicalreagent.

Accordingly, another aspect of the present invention contemplates amethod for detecting an agent which exhibits inhibitory activity to anHBV, exhibiting resistance or decreased sensitivity to TDF and/or LMV,or nucleoside analog other than TDF or LMV or an immunological reagentsaid method comprising:

-   -   generating a genetic construct comprising a replication        competent-effective amount of the genome from said HBV contained        in a plasmid vector and then transfecting said cells with said        construct;    -   contacting said cells, before, during and/or after transfection,        with the agent to be tested;    -   culturing said cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to said agent; and    -   subjecting the cells, cell lysates or culture supernatant fluid        to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of said agent.

Still another aspect of the present invention provides a method fordetecting an agent which exhibits inhibitory activity to an HBV,exhibiting resistance or decreased sensitivity to TDF and/or LMV, ornucleoside analog other than TDF or LMV or an immunological reagent saidmethod comprising:

-   -   generating a genetic construct comprising a replication        competent-effective amount of the genome from said HBV contained        in or fused to an amount of a baculovirus genome effective to        infect cells and then infecting said cells with said construct;    -   contacting said cells, before, during and/or after infection,        with the agent to be tested;    -   culturing said cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to said agent; and    -   subjecting the cells, cell lysates or culture supernatant fluid        to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of said agent.

Still another aspect of the present invention provides a method fordetecting an agent which exhibits inhibitory activity to an HBV,exhibiting resistance or decreased sensitivity to TDF and/or LMV, ornucleoside analog other than TDF or LMV or an immunological reagent saidmethod comprising:

-   -   generating a genetic construct comprising a replication        competent-effective amount of the genome from said HBV contained        in or fused to an amount of a baculovirus genome effective to        infect cells and then infecting said cells with said construct;    -   contacting said cells, before, during and/or after infection,        with the agent to be tested;    -   culturing said cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to said agent; and    -   subjecting the cells, cell lysates or culture supernatant fluid        to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of said agent.

Preferably, the HBV genome is stably integrated into the cells' genome.

Whilst the baculovirus vector is a particularly useful in the practiceof the present invention, the subject invention extends to a range ofother vectors such as but not limited to adenoviral vectors.

The present invention further extends to cell lines carrying geneticconstructs comprising all or a portion of an HBV genome or a gene orpart of a gene therefrom.

The present invention also provides for the use of the subject HBVvariants to screen for anti-viral agents. These anti-viral agentsinhibit the virus. The term “inhibit” includes antagonizing or otherwisepreventing infection, replication, assembly and/or release or anyintermediate step. Preferred anti-viral agents include nucleosideanalogs, however, the present invention extends tonon-nucleoside/non-nucleotide analog molecules.

In addition, rational drug design is also contemplated to identify orgenerate chemical molecules which either mimic a nucleoside ornucleotide analog or other DNA polymerase antagonist or which interactwith a particular nucleotide sequence or a particular nucleotide.Combinatorial chemistry and two hybrid screening are some of a number oftechniques which can be employed to identify potential therapeutic ordiagnostic agents.

In one example, the crystal structure of polymerase is used torationally design small chemical molecules likely to interact with keyregions of the molecule required for function and/or antigenicity. Suchagents may be useful as inhibitors of polymerase activity.

Several models of the HBV polymerase have been prepared due to thesimilarity with reverse transcriptase from HIV (Das et al., J. Virol.75(10): 4771-4779, 2001; Bartholomeusz et al., Antivir Ther. 9:149-60,2004; Allen et al., Hepatology 27(6): 1670-1677, 1998). The models ofthe HBV polymerase can be used for the rational drug design of newagents effective against HBV encoding the resistant mutations as well aswild type virus. The rational drug that is designed may be based on amodification of an existing antiviral agent such as the agent used inthe selection of the HBV encoding the mutations associated withresistance. Viruses or clones expressing HBV genomic material encodingthe mutations may also be used to screen for new antiviral agents.

The above methods are particularly useful in identifying an inhibitor ofa TDF— and/or LMV-resistant HBV. The present invention extends,therefore, to compositions of the inhibitors. The inhibitors may also bein the form of antibodies or genetic molecules such as ribozymes,antisense molecules and/or sense molecules for co-suppression or theinduction of RNAi.

The term “composition” includes a “pharmaceutical composition”.

The inhibitor is referred to below as an “active ingredient” or “activecompound” and may be selected from the list of inhibitors given above.

The composition may include an antigenic component of the HBV, adefective HBV variant or an agent identified through natural productscreening or rational drug design (including combinatorial chemistry).

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The pharmaceutical composition may also comprise genetic molecules suchas a vector capable of transfecting target cells where the vectorcarries a nucleic acid molecule capable of encoding an aspartyl proteaseinhibitor. The vector may, for example, be a viral vector.

Pharmaceutical forms suitable for injectable use include sterile aqueoussolutions (where water soluble) and sterile powders for theextemporaneous preparation of sterile injectable solutions. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dilution mediumcomprising, for example, water, ethanol, polyol (for example, glycerol,propylene glycol and liquid polyethylene glycol, and the like), suitablemixtures thereof and vegetable oils. The proper fluidity can bemaintained, for example, by the use of superfactants. The preventions ofthe action of microorganisms can be brought about by variousanti-bacterial and anti-fungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminium monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with theactive ingredient and optionally other active ingredients as required,followed by filtered sterilization or other appropriate means ofsterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, suitable methods of preparation includevacuum drying and the freeze-drying technique which yield a powder ofactive ingredient plus any additionally desired ingredient.

When the active ingredient is suitably protected, it may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets. For oral therapeuticadministration, the active ingredient may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers and the like.Such compositions and preparations should contain at least 1% by weightof active compound. The percentage of the compositions and preparationsmay, of course, b varied and may conveniently be between about 5 toabout 80% of the weight of the unit. The amount of active compound insuch therapeutically useful compositions is such that a suitable dosagewill be obtained. Preferred compositions or preparations according tothe present invention are prepared so that an oral dosage unit formcontains between about 0.1 μg and 300 mg of active compound (TDF isadministered at 300 mg single dose). Alternative dosage amounts includefrom about 1 μg to about 1000 mg and from about 10 μg to about 500 mg.These dosages may be per individual or per kg body weight.Administration may be per hour, day, week, month or year.

The tablets, troches, pills, capsules and the like may also contain thecomponents as listed hereafter. A binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavoringagent such as peppermint, oil of wintergreen or cherry flavoring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and a flavoring. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompound(s) may be incorporated into sustained-release preparations andformulations.

As stated above, the present invention further extends to an isolatedHBsAg from the HBV variants herein described. More particularly, thepresent invention provides an HBsAg or a recombinant form thereof orderivative or chemical equivalent thereof. The isolated surfacecomponent and, more particularly, isolated surface antigen or itsrecombinant, derivative or chemical equivalents are useful in thedevelopment of biological compositions such as vaccine formulations.

Yet another aspect of the present invention provides a compositioncomprising a variant HBV resistant to TDF and/or LMV and optionallyother nucleoside analogs or an HBV surface antigen from said variant HBVor a recombinant or derivative form thereof or its chemical equivalentand one or more pharmaceutically acceptable carriers and/or diluents.

As indicated above, antibodies may be generated to the mutant HBV agentsand used for passive or direct vaccination against infection by theseviruses. The antibodies may be generated in humans or non-human animals.In the case of the latter, the non-human antibodies may need to bedeimmunized or more specifically humanized prior to use. Deimmunized mayinclude, for example, grafting complimentary determining regions (CDRs)from the variable region of a murine or non-human animal anti-HBVantibody onto a human consensus fragment antibody binding (Fab)polypeptide. Alternatively, amino acids defining epitopes in thevariable region of the antibody may be mutated so that the epitopes areno longer recognized by the human MHC II complex.

Insofar as ribozyme, antisense or co-suppression (RNAi) repression isconcerned, this is conveniently aimed at post-transcription genesilencing. DNA or RNA may be administered or a complex comprising RNAior a chemical analog thereof specific for HBV mRNA may be employed.

All such molecules may be incorporated into pharmaceutical compositions.

Generally, if an HBV is used, it is first attenuated. The biologicalcomposition according to this aspect of the present invention generallyfurther comprises one or more pharmaceutically acceptable carriersand/or diluents.

The present invention extends to vaccines. For example, the vaccine maycomprise (1) an HBV variant as described herein alone; (2) the viralvariant in combination with an anti-HBV agent; and/or (3) an agentcapable of inhibiting the subject HBV variants.

The present invention is further directed to the use of defective HBVvariants in the manufacture of therapeutic vaccines to vaccinateindividuals against infection by HBV strains having a particularnucleotide sequence or encoding a particular polymerase.

The subject invention extends to kits for assays for variant HBVresistant to TDF and/or LMV. Such kits may, for example, contain thereagents from PCR or other nucleic acid hybridization technology orreagents for immunologically based detection techniques. A particularlyuseful assay includes the reagents and components required forimmobilized oligonucleotide- or oligopeptide-mediated detection systems.

Still another aspect of the present invention contemplates a method fordetermining the potential for an HBV to exhibit reduced sensitivity toTDF and/or LMV or optionally other nucleoside analogs, said methodcomprising isolating DNA or corresponding mRNA from said HBV andscreening for a mutation in the nucleotide sequence encoding HBV DNApolymerase resulting in at least one amino acid substitution, deletionand/or addition in any one or more of domains F and A through E or aregion proximal thereto of said DNA polymerase and associated withresistance or decreased sensitivity to TDF and/or LMV wherein thepresence of such a mutation is an indication of the likelihood ofresistance to said TDF and/or LMV.

An assessment of a potential viral variant is important for selection ofan appropriate therapeutic protocol. Such an assessment is suitablyfacilitated with the assistance of a computer programmed with software,which inter alia adds index values (I_(V)s) for at least two featuresassociated with the viral variants to provide a potency value (P_(A))corresponding to the resistance or sensitivity of a viral variant to aparticular chemical compound or immunological agent. The I_(V)s can beselected from (a) the ability to exhibit resistance for reducedsensitivity to a particular compound or immunological agent; (b) analtered DNA polymerase from wild-type HBV; (c) an altered surfaceantigen from wild-type HBV; or (d) morbidity or recovery potential of apatient. Thus, in accordance with the present invention, I_(V)s for suchfeatures are stored in a machine-readable storage medium, which iscapable of processing the data to provide a P_(A) for a particular viralvariant or a biological specimen comprising same.

Thus, in another aspect, the invention contemplates a computer programproduct for assessing the likely usefulness of a viral variant orbiological sample comprising same for determining an appropriatetherapeutic protocol in a subject, said product comprising:

-   (1) code that receives as input I_(V)s for at least two features    associated with said viral agents or biological sample comprising    same, wherein said features are selected from:    -   (a) the ability to exhibit resistance for reduced sensitivity to        a particular compound or immunological agent;    -   (b) an altered DNA polymerase from wild-type HBV;    -   (c) an altered surface antigen from wild-type HBV; or    -   (d) morbidity or recovery potential of a patient;    -   (e) altered replication capacity (increased or decreased);-   (2) code that adds said IVs to provide a sum corresponding to a    P_(V) for said viral variants or biological samples; and-   (3) a computer readable medium that stores the codes.

In a related aspect, the invention extends to a computer for assessingthe likely usefulness of a viral variant or biological sample comprisingsame in a subject, wherein said computer comprises:

-   (1) a machine-readable data storage medium comprising a data storage    material encoded with machine-readable data, wherein said    machine-readable data comprise I_(V)s for at least two features    associated with said viral variant or biological sample; wherein    said features are selected from:—    -   (a) the ability to exhibit resistance for reduced sensitivity to        a particular compound or immunological agent;    -   (b) an altered DNA polymerase from wild-type HBV;    -   (c) an altered surface antigen from wild-type HBV; or    -   (d) morbidity or recovery potential of a patient;    -   (e) altered replication capacity (increased or decreased);-   (2) a working memory for storing instructions for processing said    machine-readable data;-   (3) a central-processing unit coupled to said working memory and to    said machine-readable data storage medium, for processing said    machine readable data to provide a sum of said I_(V)s corresponding    to a P_(V) for said compound(s); and-   (4) an output hardware coupled to said central processing unit, for    receiving said P_(V).

Any general or special purpose computer system is contemplated by thepresent invention and includes a processor in electrical communicationwith both a memory and at least one input/output device, such as aterminal. Such a system may include, but is not limited, to personalcomputers, work stations or mainframes. The processor may be a generalpurpose processor or microprocessor or a specialised processor executingprograms located in RAM memory. The programs may be placed in RAM from astorage device, such as a disk or pre-programmed ROM memory. The RAMmemory in one embodiment is used both for data storage and programexecution. The computer system also embraces systems where the processorand memory reside in different physical entities but which are inelectrical communication by means of a network. For example, a computersystem having the overall characteristics set forth in FIG. 2 may beuseful in the practice of the instant invention. More specifically, FIG.2 is a schematic representation of a typical computer work stationhaving in electrical communication (100) with one another via, forexample, an internal bus or external network, a processor (101), a RAM(102), a ROM (103), a terminal (104), and optionally an external storagedevice, for example, a diskette, CD ROM, or magnetic tape (105).

The present invention further provides a kit.

In one embodiment, the present invention provides a kit for an assay forvariant HBV resistant to TDF, LMV, a nucleoside analog other than TDF orLMV or an immunological reagent said kit comprising the reagents fromPCR or other nucleic acid hybridization technology or reagents forimmunologically based detection techniques.

The present invention is further described by the following non-limitingExamples.

Example 1 Overlapping Genome of HBV

The overlapping genome of HBV is represented in FIG. 1. The geneencoding DNA polymerase (P), overlaps the viral envelope genes, Pre-S1and Pre-S2, and partially overlaps the X and core (C) genes. The HBVenvelope comprises small, middle and large HBV surface antigens. Thelarge protein component is referred to as the HBV surface antigen(HBsAg) and is enclosed by the S gene sequence. The Pre-S1 and Pre-S2gene sequences encode the other envelope components.

Example 2 Patient and Treatment

A total of 20 (47%) individuals were infected with WT HBV RT despiteprolonged exposure to LMV and TDF, whilst of the remaining 23 (53%), twoselected de novo mutations known to be associated with LMV resistance,19 showed the same LMV-associated mutations already present at baseline,and 2 patients reverted to WT. Overall, 6 distinct LMV mutationalpatterns were observed (see Table 3) rtL180M+rtM204V,rtL180M+rtM204V+rtV207M/I, rtV173L+rtL180M+rtM204V, rtL180M+rtM204I,rtM204I and rtV191I.

Two patients selected viruses with novel mutations (see Table 3), bothwere HBeAg positive and genotype A.

TABLE 3 Mutational patterns of the HBV pol in 23 HBV/HIV-coinfectedindividuals exposed to tenofovir for longer than 6 months. Pattern No.of patients Mutational pattern A 10 rtL180M + rtM204V B 4 rtL180M +rtM204V + V207M/I C 4 rtV173L + rtL180M + rtM204V D 1 rtL180M + rtM204IE 1 rM204I F 1 rtV191I G 1 rtA194T + rtL180M + rtM204V H 1 rtA194T +rtV173L + rtL180M + rtM204V Patterns A-F correspond to lamivudineresistance while patterns G-H correspond to novel mutational patterns.Italic represents the novel amino acid change.

Patient A:

31 year old male, HIV+, HBsAg+, was treatment at Hospital Carlos III,Madrid, with DDI, TDF+LPV/r. After 36 weeks therapy changed to AZT, 3TC,TDP+LPV/r. After commencing treatment, plasma HIV RNA remained belowdetectable levels (<50 cop/mL) and absolute CD4 counts steadilyincreased from 224 to 594 cel/uL (% from 7% to 22%). The patient isinfected with HBV genotype A, positive for HBeAg, antiHBc, and negativefor antiHBs, antiHBc IgM. Plasma HBV DNA and ALT were measured every 3-4months. HBV DNA was measured using Roche COBAS Monitor (see Table 4).

TABLE 4 The HIV and HBV DNA viral load and Liver function test (alaninetransaminase; ALT) for Patient A HIV HBV ALT week (cop/mL) (cop/mL)(IU/mL) 79 <200 <50 56 62 300 <50 41 48 1000 <50 212 36 700 <50 29 1613000 <50 46 0 <50 41 −8 34966 61

Patient B:

44 year old male, HIV+, HBsAg+, HAV+, started treatment in 1995 with AZTand DDC (see Table 4 for AntiRetroviral therapy history). Patient Bpresented with mutations in the HBV RT of rtV173L, rtA194T, rtL180M andrtM204V, 77 weeks after the start of TDF therapy. He had initiated histreatment with zidovudine and zalcitabine in 1995. Treatment with TDFalong with LMV and lopinavir boosted with ritonavir was prescribed in2002. After 8 weeks, the patient voluntarily stopped therapy for 37weeks. Antiretroviral treatment was resumed with TDF, LMV and efavirenz.Plasma HBV-DNA decreased from 7.2 log copies/ml to 1.7 log copies/ml in64 weeks after restart of ART. During this period, ALT levels remainedconstantly normal and CD4+ counts ranged from 315 to 553 cells/μl,always with undetectable plasma HIV-RNA levels during treatment (FIG. 17b).

TABLE 5 Antiviral History for Patient B HAART Week AZT/ddC −338 ddI/3TC−308 ddI/3TC/IDV −286 D4T/ddI/NVP −142 D4T/3TC/ABC −111 D4T/ABC/APVr−100 D4T/APVr −96 D4T/3TC/LPVr −49 ddI/3TC/LPVr −45 3TC/LPVr −1TDF/3TC/LPVr 0 No treatment 33 TDF/3TC/EFV 46

After treatment with TDF, HIV RNA, HBV DNA and ALT were measured andlevels are listed in Table 6. Absolute CD4 counts ranged from 324 to 602cel/uL (mean 457 cel/uL) (mean CD4%; 13%). The patient was infected withHBV genotype A, and was HBeAg+.

TABLE 6 HIV RNA, HBV DNA and ALT Levels HIV HBV ALT week (cop/mL)(cop/mL) (IU/mL) 116 50 48 N.D. 101 50 84 18 89 50 479 23 77 50 3840 2657 50 39700 30 52 50 18600000 26 42 5120 200000 37 33 2430 7440 40 −2 503.49E+11

Example 3 Detection of Viral Markers

Hepatitis B surface antigen (HBsAg), hepatitis Be antigen (HBeAg),anti-HBe and hepatitis B core antigen (HBcAg) specific IgG and IgM weremeasured using commercially available immunoassays. Hepatitis B viralDNA levels were measured using Roche COBAS Monitor assay according tothe manufacturer's directions.

Example 4 Sequencing of HBV DNA

HBV DNA was extracted and sequenced using standard published methods.The following primers were used for amplification and sequencing:

(SEQ ID NO: 1) (5′-3′) Primer 1 forward: TCTTGTTGACAAGAATCCTCAC, (SEQ IDNO: 2) (5′-3′) Primer 2 forward: AGACTCGTGGTGGACTTCTCT (SEQ ID NO: 3)(5′-3′) Primer 3 reverse: CCCAAAAGACCCACAATTC, and (SEQ ID NO: 4)(5′-3′) Primer 4 reverse: TGACATACTTTCCAATCAAT.

Both 1st and 2nd round PCR conditions were as follows: 2 min 95 C, (30sec 95 C, 30 sec 50 C, 90 sec 72 C)×30 cycles, 72 C for 4 mins.

Example 5 Analysis of HBV DNA Patient A:

At week 48 the patient had no mutations associated to resistance nor anynovel amino-acid changes. However, at week 62 the patient selectedmutations rtL180M and rtM204V as well as a novel mutation rtA194T. SeeTable 7.

Patient B:

No sera for sequence analysis was available before the treatment withTDF commenced, but at week 77 the patient presented mutations rtV173L,rtL180M and rtM204V together with novel mutation rtA194T. The HBsAgmutations included sE164D, sS193L and sI195M. See Table 8.

TABLE 7 Summary of HBV mutations in patient A treated with TDF and LMVSample name Genotype Polymerase* Surface 0 A rtS54T — 48 A rtS54T — 62 ArtL180M sI195M rtA194T rtM204V Nomenclature according to Stuyver et al.,2001, supra

TABLE 8 Summary of HBV mutations in patient B treated with TDF and LMVSample name Genotype Polymerase* Surface 77 A rtV173L sE164D rtL180MsS193L rtA194T sI195M rtM204V

Example 6 Modelling of the HBV Polymerase with rtA194T

The HBV polymerase has not been crystallized and a homology model basedon similarity with HIV has been developed (Bartholomeusz et al, 2004supra). Using this model mutations were located at rtA194T.

The HBV reverse transcriptase mutation rtA194T is located away from theactive site towards the end of the alpha helix in a potential hingeregion which contains the B domain (FIG. 15).

Two potential mechanisms by which the mutation at rtA194T may influenceresistance to TDF and/or LMV.

-   -   1. The mutation at rtA194T in the hinge region may still have        the ability to affect the position alpha helix that encodes the        B domain. This is where codon rt180 is located, and this region        has the potential to interact with the DNA template. Changes to        this region may alter the position of the DNA template strand        relative to the dNTP binding site.    -   2. The reverse transcriptase (rt) region is only part of the        polymerase protein. The entire HBV polymerase is a large protein        (90 kd). The mutation is on the external surface of the rt        region. There is no structural homologues for the terminal        protein and spacer region components of the HBV polymerase        protein that also includes an RNase H component. Therefore, the        mutation has the potential to interact with the rest of        components the polymerase protein. This has been demonstrated in        HIV in which mutations which affect the RNAse H component have        been known to also alter antiviral sensitivity.

Example 7 TDF

TDF is a potent inhibitor of HBV replication. TDF has activity againsthepadnaviruses and HIV. The structure of TDF is shown in FIG. 16 and itssynthesis is described in a review by Grim S A, Romanelli F, AnnPharmacother 37(6):849-59, 2003. Preclinical studies indicate that TDFis a highly potent inhibitor of HBV in enzyme- and cell-based assays

Example 8 Replication Competent HBV Plasmids

An extended (1.28×) HBV genome was cloned (HBV genotype A, subtype adw2)into a pBluescript KS(+) plasmid (Stratagene, La Jolla, Calif., USA), asdescribed by Tacke et al, J Virol 78:8524-8535, 2004.

Example 9 Site-Directed Mutagenesis

Three mutated HBV plasmids were constructed using a Quikchange(Registered) II site-directed mutagenesis kit (Stratagene, La Jolla,Calif., USA). Plasmids contained either mutations rtL180M and rtM204V(LM), rtL180M, rtM204V and rtA194T (LMAT), or rtA194T (AT). To createLAM-specific mutations rtL180M and rtM204V, primers rtM204VF (CTT TCAGCT ATG TGG ATG ATG TGG TAT TGG (SEQ ID NO:17)) and rtM204VR (CCA ATACCA CAT CAT CCA CAT AGC TGA AAG (SEQ ID NO:18)), and rtL180MF (TCC GTTTCT CAT GGC TCA GTT TAC TAG TG (SEQ ID NO:19)) and rtL180MR (CAC TAG TAAACT GAG CCA TGA GAA ACG GA (SEQ ID NO:20)), respectively, were designed.Mutations were created according to Bock et al, Gastroenterology122:264-273, 2002.

Mutation rtA194T was engineered using mutagenic primers A194TR (AGT GGTTCG TAG GAC TTT CCC CCA CTG TTT GG (SEQ ID NO:21)) and A194TF (AGC CAAACA GTG GGG GAA AGT CCT ACG AAC CAC (SEQ ID NO:22)). All plasmids wereultimately sequenced to verify that only the intended mutations had beenintroduced.

Example 10 Cell Culture and Viral Transfection

HepG2 cells were grown in Eagles minimum essential medium (LGCPromochem, Middlesex, UK), supplemented with 10% v/v fetal bovine serumat 37° C. and 5% v/v CO₂. For cDNA transfections, 3×10⁵ cells wereseeded to semi-confluence onto each well of a 6-well plate and allowedto adhere overnight. The following day, 950 ng of HBV cDNA plasmid wasco-transfected with 50 ng of pCMV β-Galactosidase vector (Promega,Mannheim, Germany) into each well, using the Fugene6 transfection agentand following the manufacturer's instructions (Roche, Indianapolis,USA). Five hours after transfection, cells were washed with PBS and theculture medium was replaced with media containing TDF at concentrationsfrom 0 to 100 μM. Seventy-two hours after transfection, the cells werewashed and the media changed again with the appropriate drugconcentrations Media and cell lysates were harvested for analysis 5 daysafter transfection. Transfection efficiency was controlled by measuringβ-galactosidase activity from cell lysates according to manufacturer'sinstructions (Promega).

Example 11 Quantification of Extracellular HBV-DNA

HBV viral particles from the cell culture supernatant were precipitatedusing 26% polyethylene glycol (PEG 8000, Sigma, St. Louis, Mo., USA),followed by DNase 1 digestion (Takara, Shiga, Japan). DNA was extractedusing a QiaAMP viral DNA extraction kit (Qiagen). A quantitativereal-time PCR was used to quantify HBV-DNA. Briefly, a 189-bp fragmentof the X region was amplified in a LightCycler system (Roche), using theQuantiTect Probe PCR kit (Qiagen) and the following probes and primers:3FL-XHybprobe (5′-ACG GGG CGC ACC TCT CTT TAC GCG G fluorescein-3′ (SEQID NO:23)), 5LC-Xhybprobe (5′LCRed 640 nm-CTC CCC GTC TGT GCC TTC TCATCT GC PH-3′ (SEQ ID NO:24)), X sense (5′-GAC GTC CTT TGT YTA CGT CCCGTC-3′ (SEQ ID NO:25)), and X antisense (5′-TGC AGA GGT GAA GCG AAG TGCACA-3′ (SEQ ID NO:26)). The LightCycler program consisted of an initialactivation step (95° C., 15 min, slope 20° C./sec), 40 cycles ofamplification with touch down (95° C. 10 sec, 62-55° C. 15 sec, 72° C.13 sec, slope of 5° C./sec), melting curve (cooling from 95° C. to 50°C. for 10 sec with a slope of 20° C./sec and heating again to 95° C. for0 sec with a slope of 0.1° C./sec), and cooling (40° C. for 30 sec witha slope of 20° C./sec). Standard dilutions of a plasmid containing theHBV X gene (X-pScript, Stratagene) were used to generate a standardcurve. All measurements were carried out in triplicate.

After adjusting for transfection efficiency, IC₅₀ values were calculatedfrom the HBV viral load using the following formula (Vandamme et al,Antiviral methods and protocols:231, 2000):

log IC₅₀=log conc.HPP−[(HPP−50)/(HPP−LPP)×log d

HPP, highest protective percentage; LPP, lowest protective percentage;d, dilution.

Example 12 Toxicity Determination

The toxicity of TDF was measured by transfecting HepG2 cells in 96-wellsplates and continually exposing the test drugs up to 10 mM for 5 days.MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] wasadded to each well at a final concentration of 500 μg/mL five hoursbefore dissolving crystals in 200 uL of DMSO and measuring at 550 nm UVwavelengths.

Example 13 Phenotypic Analyses

Following the genetic analysis, further analyses of the novel mutationsobserved in the two patients described above were undertaken. As theclinical data suggested a particular relevance of the novel rtA194Tmutation, especially in combination with LMV resistance, site-directedmutagenesis was performed and 3 replication-competent HBV plasmidscarrying the following mutations: rtL180M+rtM204V (LM),rtL180M+rtA194T+rtM204V (LMAT), and rtA194T (AT) were constructed. Theantiviral activity of TDF was examined in a transient transfection cellculture assay system along side a WT construct as described above.

Extracellular HBV-DNA was examined by real-time PCR, 5 dayspost-transfection and IC₅₀ values were calculated. The results aresummarized in Table 9, which shows the resistance profile for the mutantHBV.

TABLE 9 IC₅₀ values for tenofovir obtained from transient transfectioncell culture assays using WT and mutant HBV-DNA constructs transfectedinto HepG2 cells. Effect of clinical mutations on HBV susceptibility totenofovir in vitro. Extracellular DNA IC₅₀ (μmol/L) Fold IC₅₀ WT   12.41    A194T (AT) 95 7.6^(a) L180M + M204V (LM) 71 5.7^(a) L180M, A194T,M204V (LMAT) >120*   >10*^(b)   *IC₅₀ values above the cell toxic levelof tenofovir. ^(a)5-10 fold increase in the IC₅₀ confers to partialresistance [24] ^(b)>10-fold increase in the IC₅₀ confers to resistance[24]

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

-   Allen et al., Hepatology 27(6): 1670-1677, 1998-   Aye et al., J Hepatol. 26: 1148-53, 1997-   Bartholomeusz et al., Antivir Ther. 9(2):149-160, 2004-   Bock et al, Gastroenterology 122:264-273, 2002-   Boyd et al., Antiviral Chem Chemother. 32: 358-363, 1987-   Das et al., J. Virol. 75(10): 4771-4779, 2001-   Delaney et al., Antimicrob Agents Chemother 45(6): 1705-1013, 2001-   Dienstag et al., New England J Med 333: 1657-1661, 1995-   Gaillard et al., Antimicrob Agents Chemother. 46(4): 1005-1013, 2002-   Grim S A, Romanelli F, Ann Pharmacother.; 37(6):849-59, 2003-   Kruger et al., Hepatology 22: 219A, 1994-   Main et al., J Viral Hepatitis 3: 211-215, 1996-   Lada O et al., Antivir Ther.; 9:353-63, 2004-   Norder et al., J. Gen. Virol. 74: 341-1348, 1993-   Ren and Nassal, J. Virol. 75(3): 1104-1116, 2001-   Severini et al., Antimicrobial Agents Chemother 39: 1430-1435, 1995-   Shaw J P et al., Pharm Res. 14(12):1824-9, 1997-   Stuyver et al., Hepatology 33: 751-757, 2001-   Summers and Mason, Cell 29: 403-415, 1982-   Tacke et al, J Virol 78:8524-8535, 2004-   Vandamme et al, Antiviral methods and protocols:231, 2000-   Vere Hodge, Antiviral Chem Chemother 4: 67-84, 1993-   Ying C et al., J Viral Hepat. 7(2):161-5, 2000-   Xiong et al., Hepatology. 28(6): 1669-73, 1998

1. An isolated Hepatitis B virus (HBV) variant wherein said variantcomprises nucleotide mutations in a gene encoding a DNA polymeraseresulting in the amino acid substitution mutationrtL180M+rtA194T+rtM204V in said DNA polymerase and wherein said variantexhibits decreased sensitivity to TDF.
 2. The isolated HBV variant ofclaim 1 wherein said variant further exhibits reduced sensitivity toLMV.
 3. The isolated HBV variant of claim 1 wherein the variant furthercomprises an rtV173L amino acid substitution mutation in the DNApolymerase.
 4. The isolated HBV variant of claim 1 wherein the variantfurther exhibits decreased sensitivity to an antibody to a surfacecomponent of HBV.
 5. The isolated HBV variant of claim 4 wherein thesurface component is the hepatitis B surface antigen (HBsAg).
 6. Theisolated HBV variant of claim 5 wherein the variant comprises the aminoacid substitution mutation sS193L in said HBsAg.
 7. The isolated HBVvariant of claim 6 further comprising an sE164D amino acid substitutionmutation in said HBsAg
 8. The isolated HBV variant of claim 6 furthercomprising an sI195M substitution mutation in said HBsAg.
 9. A methodfor determining the potential for an HBV to exhibit reduced sensitivityto TDF and/or LMV said method comprising isolating DNA or correspondingmRNA from said HBV and screening for mutations in the nucleotidesequence encoding HBV DNA polymerase resulting in the amino acidsubstitution rtL180M+rtA194T+rtM204V in said DNA polymerase wherein thepresence of such a mutation is indicative of reduced sensitivity to TDFand/or LMV.
 10. The method of claim 8 further comprising identifying anrtV173L amino acid substitution mutation in the DNA polymerase.
 11. Amethod for determining the potential for an HBV to exhibit reducedsensitivity to TDF and/or LMV said method comprising isolating DNA orcorresponding mRNA from said HBV and screening for the substitutionmutation sS193L in HBsAg wherein the presence of such a mutation isindicative of reduced sensitivity to TDF and/or LMV.
 12. The method ofclaim 11 further comprising screening for the presence of an sE164Dsubstitution in said HBsAg.
 13. The method of claim 11 furthercomprising screening for the presence of an SI195M substitution in thesaid HBsAg.
 14. A method for detecting an agent which exhibitsinhibitory activity to an HBV which exhibits resistance or decreasedsensitivity to TDF and/or LMV said method comprising: generating agenetic construct comprising a replication competent-effective amount ofthe genome from an HBV which carries the substitution mutation in itsDNA polymerase rtL180M+rtA194T+rtM204V contained in a plasmid vector andthen transfecting said cells with said construct; contacting said cells,before, during and/or after transfection, with TDF and/or LMV to betested; culturing said cells for a time and under conditions sufficientfor the HBV to replicate, express genetic sequences and/or assembleand/or release virus or virus-like particles if resistant to TDF and/orLMV; and subjecting the cells, cell lysates or culture supernatant fluidto viral- or viral-component-detection means to determine whether or notthe virus has replicated, expressed genetic material and/or assembledand/or been released in the presence of TDF and/or LMV.
 15. The methodof claim 14 wherein the HBV carriers a further rtV173L substitution inthe DNA polymerase.
 16. The method of claim 14 wherein the HBV carriersa further sS193L substitution mutation in the gene encoding HBsAg 17.The method of claim 14 wherein the HBV carries a further sE164D and/orsI195M substitution in the gene encoding HBsAg.
 18. A computer productfor assessing the likely sensitivity to an HBV to TDF and/or LMV, saidproduct comprising: (1) code that receives as input code for at leastone feature associated with said viral agents wherein said features is asubstitution mutation in a DNA polymerase which isrtL180M+rtA194T+rtM204V; (2) a computer readable medium that stores andrecords the codes; and (3) an output hardware for receiving andreporting the presence of a mutation associated with resistance to TDFand/or LMV.
 19. The computer product of claim 18 further comprising afeature which is an rtV173L substitution mutation in the DNA polymerase.20. A method for the treatment or prophylaxis of HBV infection in amammal, comprising administering an HBV variant resistant to TDF and/orLMV as defined in claim 1 to the mammal.
 21. A vaccine comprising anisolated HBV of claim 1 or an antigenic component thereof and one ormore pharmaceutically acceptable carriers and/or diluents.